The long-term goal of this research is to elucidate the mechanism of muscle contraction. This grant will support work on three projects that focus on critical aspects of actomyosin function and its regulation in the cross-bridge cycle. The following specific aims will be pursued in the three projects: I. Strongly Bound Acto-S1 States: (i) to test the hypothesis for a specific role of loop 2 on myosin heads (S1) and actin's N-terminus in the transition from the weakly to strongly bound acto-S1 states; (ii) to test the role of sequence 50-58 on actin in acto-S1 interactions; (iii) to test and refine the models of the acto-S1 structure by mutations to cysteine on actin and S1, and the subsequent cross-linking (disulfide and other) and mapping of the acto-S1 interface. II. Dynamic Transitions and Couplings in S1: (i) to map the conformational states of the S1 relay region in the nucleotide and actin bound states of S1; (ii) to compare the flexibility of the SH1-SH2 helix in scallop and skeletal S1 by modifications and cross-linking of their SH1 and SH2 groups; (iii) to test and map several coupling pathways in S1: between the SH1-SH2 helix, the relay region, the converter, loops 1 and 2, and other sites on S1. III. Structure, Dynamics and Regulation of F-actin: (i) to test and refine the models of F- actin structure and the intermolecular interfaces in F-actin through disulfide cross-linkings of cysteine yeast actin mutants and the subsequent model calculations; (ii) to test the role of the hydrophobic plug (residues 262-274) in actin polymerization; (iii) to crystallize cross-linked (Q41-C374) alpha-skeletal actin dimers bound to segment 1 of gelsolin; (iv) to document the role of dynamic changes on F-actin in its function and regulation. The proposed research will combine the powerful approach of mutational substitutions in proteins with solution measurements of actomyosin function (acto-S1 binding, ATPase activity, regulation, in vitro motility, and force generation) and with the studies of F-actin, S1, and acto-S1 structure and dynamics through labeling, cross-linking, fluorescence measurements, and computational modeling. Key components of the proposed research are the preparation of mutant proteins, and the collaborative integration of the work on myosin and actin. The results of this work will contribute to the understanding of muscle function and malfunction, especially in the growing number of genetically identified cardiac and skeletal muscle myopathies.